Through comparative studies between distantly related animals, it has become apparent that genes and regulatory networks functioning during embryonic growth are highly conserved. This has led to the hypothesis that evolutionary changes in morphology can be traced to alterations in these regulatory modules. Studies to address this hypothesis have focused on insects, which display different modes of segmentation. Most approaches rely on cloning and expression analysis of orthologs of well-characterized Drosophila genes. However, most insects do not offer facile approaches to examine the functional significance of conserved gene expression patterns, or to test observed differences. Further, these comparisons are limited to the analysis of mechanisms discovered in flies, and do not offer the possibility of identifying genes important to segmentation in species other than flies. Our studies in Tribolium overcome these limitations, since Tribolium offers the possibility of genetic manipulation in addition to its facility for developmental and molecular studies. Moreover, the recent advances in RNA interference and germline transformation place Tribolium in the forefront of comparative model systems. We have discovered that depletion of certain pair-rule gene mRNAs by RNAi blocks segmentation and morphogenesis in Tribolium, results not predicted by the Drosophila paradigm. To understand the molecular interaction underlying these novel phenotypes we will examine the effects of Tceve and Tcrun mRNA depletion on the expression of other segmentation and homeotic genes. Analysis of the regulatory regions associated with these genes and ectopic expression of transgenes will complement the RNAi studies. To discover other genes important to segmentation in Tribolium we will execute a transposon-tagging mutagenesis scheme and characterize relevant mutants. Our research provides a unique opportunity to elucidate the genetic mechanisms underlying the regulation of the process of progressive segmentation in a cellular environment.